Cavity induced allosteric modification of intermolecular interactions and methods of identifying compounds that effect the same

ABSTRACT

Method of identifying compounds that modulate intermolecular interactions between a target protein and a modifier are disclosed. Pharmaceutical composition comprising compounds that inhibit intermolecular interactions between a target protein and a modifier are disclosed. Methods of treating individual suffering from inflammatory conditions, undesirable immune responses, immunological conditions and bacterial infections are disclosed.

ACKNOWLEDGMENT OF GOVERNMENT RIGHTS

The present invention was made under Grant 1R21RR13360-01 from theNational Institutes of Health. The Government may have certain rights tothe invention.

FIELD OF THE INVENTION

The present invention relates to the identification of compounds thatmodulate intermolecular interactions by allosterically modifying afunctionally critical site of a protein involved in such interactionsand to methods of identifying the same.

BACKGROUND OF THE INVENTION

This application claims priority to provisional application Ser. No.60/091,431, filed Jul. 1, 1998 and Ser. No. 60/133,435, filed May 11,1999, which both have the same title as this application and which areboth incorporated herein by reference.

One of the challenges in the development of therapeutic compounds is tofind a small molecule that is able to mediate a desired biologicaleffect. Traditionally, synthetic chemistry and natural product screeninghave been the principal means for the derivation of many drug products.

High-throughput random screening is a standard procedure adopted bypharmaceutical companies for the discovery of lead compounds. Thismethod relies upon availability of a large chemical database ofnatural/medicinal products. This procedure does not require theknowledge of principle components of biomolecules that cause disease. Inshort, it is a blind process to screen therapeutic lead compounds. Theadvantage of this approach is that it facilitates to build a largemedicinal chemical database and can be repeatedly used to screentherapeutic compounds. Unfortunately, random screening is tedious andoften requires isolation and characterization from natural extracts.Natural products are complex and include the stereochemical complexitiesinherent in their natural origin.

While high-throughput random screening procedures have been used toidentify some novel therapeutic molecules, such procedures are oftenlimited by the availability of large chemical databases. Advances incomputer technology and the understanding of protein-proteininteractions has allowed for attempts to replace the high-throughputscreening procedures with computer-aided analysis and design of novelmolecules. Such structure based approaches have reduced the time andresources to discover novel compounds.

Structure based approaches have been used to develop several inhibitorsthat are either “substrate analogs” or “allosteric” inhibitors.Allosteric effectors, in some cases, are considered superior toconventional substrate analog for reasons: (1) it is non-competitivewith natural ligand, (2) it can be effective at a lower concentration,(3) allosteric binding sites are less conserved and thereby specificityand selectivity can be enhanced, and (4) in some cases allostericeffectors can inhibit the target molecules' function by trapping it inan intermediate non-native or molten globular state.

Structure-based approaches represent a targeted pathway wheretherapeutic agents are designed towards the biomolecule responsible fordisease. There are two major approaches in the design of leadtherapeutic compounds based on the nature of the molecule. For enzymes,design of substrate analogs (from the knowledge of active site) andpeptidomimetics that has shown promise in some cases.

Substrate analogs are developed to compete with the natural substrateand occupy the active site. Thus, a potent therapeutic compound musthave high affinity, exhibit selectivity and have longer retention time.Substrate analogs are better suited for enzymes, because many receptorsand other non-enzyme molecules, such as receptors and their ligands haveno defined active site but alter biological function. In such cases, apeptide's ability to mimic a protein's local structural features is oneof the ways used to design therapeutic compounds. Substrate analoginteractions are often not reversible.

Peptidomimetics are developed both as therapeutic agents and as a probeto understand biological functions. Natural products targeting opioidand hormone receptors are historical examples of peptidomimetics becausethey validate many of the concepts invoked in rational design. Thesecompounds provide a classic example of how structurally differentnon-peptides may be from their peptide parents (lacking flexibility,amide bonds and obvious pharmacophore similarity) and how theirmodification can lead to highly selective ligands for subtypes ofreceptors for both peptide and non-peptide compounds.

Elucidation of the conformation of a peptide can provide insights aboutthe structural requirements of its binding to a receptor (Boteju, L. W.et al., 1996, J. Med. Chem., 39:4120-4; and Cho, M. J. et al., 1996,Trends in Biotechnology, 14:153-8; which are both incorporated herein byreference). A major problem, however, in structure-activity studies oflinear peptides is the large degree of flexibility, not only of theside-chain residues, but also of the peptide backbone. Substitution ofindividual amino acids followed by biological screening might reflectaffective differences on structure rather than on residues implicated inbinding. Consequently, spectroscopic studies in solution, where a rapidequilibrium between numerous conformations is likely to occur, have hadlittle impact on the design of linear peptide analogues. In contrast,constrained peptides delineate solution conformations for correlationwith receptor bound conformations. Bioactive compound design based uponconformational constrained peptide analogs representative of therecognition elements of the protein constitutes an effective approach tomimetic drug design. Constraints imposed upon peptides to lock in aparticular conformation often times emulate those imposed by thetertiary structure of protein ligands. Imposed constraints can reflectthe use of amino acids that contribute to the propensity of a particularsecondary structure such as amphipathic helical repeats.

Despite the diverse usefulness of peptidomimetics, they remain lessviable drugs due to their poor bioviability. Nevertheless, activepeptide analogues with modified bonds or side chains, provide anotherapproach in defining bioactive conformations and are valuablepharmacological probes, because generally they are more resistant toproteolytic degradation.

Protein structures have been elucidated using crystallography, NMR andmolecular modeling. The three dimensional structures of proteins reveal(1) overall folding of the molecule, (2) scaffolds: secondary structuralfeatures such as α-helix, β-sheet, (3) functional units; b-turns andloops, and (4) surfaces that include cavities, clefts, pockets andcrevices formed by the folding of amino acid chains on itself and, inthe case of multimeric protein complexes, on itself and the amino acidchains of other subunits. Cavities, clefts, pockets and crevices canaccommodate water molecules within an interior. Depending upon thenature of the amino acids which form the cavities, clefts, pockets andcrevices molecules, the interior of these structural features havespecific chemical and electrostatic properties as well as spatialdimensions.

Determination of crystal structures of proteins/receptors have provideda basic understanding of protein/receptors' function. Several receptorssuch as EGF receptors are activated either by ligands or by associationwith other erbB family of receptors. One of the hypothesis is thatconformational changes induced either by ligand or by co-receptorselicits signal transduction. Thus, it is presumed that throughallosteric mechanisms receptors can modulate signal transduction.Allosterically driven biological functions are also known both inenzymes and receptors (Ellis, J., 1997, Drug. Dev. Res., 40:193-204, andKundrot, C. E. et al., 1991, Biochem., 30:1478-1484, which are bothincorporated herein by reference). Attempts to modulate the function ofproteins/receptors have been made and often referred to as “allostericmodification or allosteric inhibitors”.

Allosteric modification is a well known technique that has been studiedin several enzymes (Iverson, L. F. et al., 1997, Protein Science,6:971-982; Ladjimi, M. M. et al., 1985, J. Mol. Biol., 186:715-724;Ozaita, A. et al., 1997, Brit. J. Pharm., 121:901-912; Tang, J. et al.,1997, Chemistry & Biol., 4:453-459; and Tijane, M. et al., 1989, FEBSLett., 245:30-34; which are each incorporated herein by reference) andreceptors (Berthold, M. et al., 1997, Neurochem. Res., 22:1023-1031;Ellis, J., 1997, Drug. Dev. Res., 40:193-204; Kolliasbaker, C. A. etal., 1997, J. Pharmco. Exp. Therap., 281:761-768; and Robichon, R. etal., 1997, Eur. J. Pharmco., 328:255-263 which are each incorporatedherein by reference). Hitherto techniques often used mutagenesis orsmall molecules identified from screening. Allosteric modifications havebeen used in enzymes to alter the enzymes' kinetics and in some casesused to develop inhibitors.

There is a need for modulators of intermolecular interactions and formethods of identifying such modulators. There is a need for inhibitorsof intermolecular interactions and for methods of identifying suchinhibitors. There is a need for enhancers of intermolecular interactionsand for methods of identifying such enhancers. Structure based liganddesign, as practiced today, requires the knowledge of cavity of knownfunctions such as active sites, or cavities identified by highthroughput (ligand binding). There is a need for a generalized approachto identify functional cavities for novel ligand design.

SUMMARY OF THE INVENTION

The present invention relates to methods of identifying compounds thatmodulate intermolecular interactions between a protein target and amodifier. Modulators may be inhibitors, i.e. compounds that inhibitintermolecular interactions, or enhancers, i.e. compounds that enhancesintermolecular interactions. According to the methods of the presentinvention, a cavity, cleft, pocket or crevice in the protein targetwhich is proximal to a functionally critical site of the target proteininvolved in intermolecular interactions with the modifier is identifiedthat may be distinct and proximal from the catalytic site. The volume ofthe cavity, cleft, pocket or crevice is calculated and its chemical andelectrostatic properties are mapped. Functional groups and compounds areidentified which can be accommodated by the cavity, cleft, pocket orcrevice. The parameters for identifying such functional groups andcompounds include size, charge and hydrophobicity/hydrophilicitycharacteristics. Compounds which contain functional groups that can beaccommodated by the cavity, cleft, pocket or crevice, includingcompounds which can be completely accommodated by the cavity, cleft,pocket or crevice, are then tested in an in vitro assay to determinewhether they modulate target-modifier interactions.

The present invention relates to pharmaceutical compositions and methodsof treating an individual suffering from an inflammatory condition.

The present invention relates to pharmaceutical compositions and methodsof treating an individual suffering from an undesirable immune responseor immunological condition are disclosed.

The present invention relates to pharmaceutical compositions and methodsof treating an individual suffering from a bacterial infection aredisclosed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict the structure of third domain of TNF receptor.FIG. 1A shows the disposition of cystine-knot loops (WP9) and a cavitynear the binding site. The portion of the molecule denoted with an arrowshows the loop that was used as a template to design peptidomimetic.FIG. 1B shows inhibition of TNFα-induced cytolysis of L929 cells by theantagonistic peptides. Absorbance obtained with 1 mg/ml of ACT-D aloneand with ACT-D and 50 pg/ml of TNFα were considered as 100% survival and100% cytotoxicity, respectively. The results indicate the means andstandard deviations derived from three independent experiments.

FIGS. 2A, 2B and 2C show a preliminary result from a small databasesearch in the third domain of TNF receptor. For clarity, only the domainof the receptor is shown. FIG. 2A depicts the WP9 cavity of TNFreceptor. FIG. 2B shows the molecule s7 forms a complex with a bindingenergy of −40 Kcal/mol without any chemical optimization. Since thiscompound is not chemically altered for maximal binding, kinetics ofligands have not been performed. FIG. 2C shows results when tested in anapoptosis assay similar to the peptidomimetics, i.e. about 20%protection at 300 μM concentration.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the term “target protein” is meant to refer to a proteinthat is involved in intermolecular interactions with a modifier. Thetarget protein may be a cellular protein or a protein that existsoutside of a cell. The target protein may be, for example but withoutlimitation to, a membrane bound protein, a cytosolic protein, a nuclearprotein, an enzyme, a cytokine, a lymphokine, a chemokine, an adhesionmolecule, a growth factor, or a receptor for such proteins. In someembodiments, the target protein is tumor necrosis factor (TNF) receptorfamily including TNF receptors, fas, CD40. gp30, and fas ligand, TNFα,CD4, β-lactamase, c-erbB2 p185 translation product, growth hormonereceptor, growth hormone, insulin receptor, insulin, IL-1 receptor,IL-1, IL-2 receptor, IL-2, epidermal growth factor receptor (EGFR), andepidermal growth factor (EGF). A target protein must have a cavity,cleft groove pocket of crevice as part of its three dimensionalstructure.

As used herein, the term “modifier” is meant to refer to a compoundwhich is involved in intermolecular interactions with a target protein.The modifier may a proteinaceous molecule such as a protein, polypeptideor peptide, or a non-proteinaceous molecule such as a sugar,polysaccharide, nucleic acid molecule, or other non-proteinaceousorganic or non-organic molecule. The term modifier may be usedinterchangeably herein with the term “ligand”. Examples of proteinaceousmodifiers include: proteins such as membrane bound proteins, cytosolicproteins, and nuclear proteins; and proteins, polypeptides and peptidessuch as proteinaceous enzyme substrates, cytokines, lymphokines,chemokines, adhesion molecules, growth factors, or receptors for suchmolecules. In some embodiments, the modifier is (TNF) receptor familyincluding TNF receptors, fas, CD40. gp30, and fas ligand, TNFα, CD4,β-lactamase, c-erbB2 p185 translation product, growth hormone receptor,growth hormone, insulin receptor, insulin, IL-1 receptor, IL-1, IL-2receptor, IL-2, epidermal growth factor receptor (EGFR), and epidermalgrowth factor (EGF).

As used herein, the term “intermolecular interactions” is meant to referto interactions that occur between and protein, which is referred to asthe target protein, and a second molecule, which is referred to as amodifier. The interactions occur at a site on the target proteinreferred to as the target protein:modifier interaction site.Intermolecular interactions include for example: association,oligomerization, binding, and conformational/structural perturbances.The intermolecular interaction between the target protein and themodifier results in some biological activity, the enhancement orinhibition of which is desirable in some circumstances. Examples ofintermolecular interactions which result in a biological activityinclude processing of substrates by enzymes, ligand induced signaltransduction, allosteric modulators, signal transduction dueoligomerization, and protein small molecule binding(antagonists/agonists).

As used herein, the term “target protein:modifier interaction site” ismeant to refer to the location on the target protein in whichinteraction between the target protein and the modifier occurs. Inexamples where the target protein is an enzyme and the modifier is anenzyme substrate, for example, the target protein:modifier interactionsite is also referred to as the catalytic site. In examples where thetarget protein is a receptor, for example, the target protein:modifierinteraction site is also referred to as the binding site. In some cases,such as when the target protein is a member of the immunoglobulinsuperfamily, the target protein:modifier interaction site may includecomplementarity determining regions (CDRs) or loops, which define theportions of the target protein which interact directly with modifier.

As used herein, the terms “cavity”, “cleft”, “pocket”, “groove” and“crevice” are used interchangeable and are meant to refer to a molecularsurface or location on a target protein that can accommodate at leastone solvent such as for example water molecules, although some cavitiesmay not be solvated. The identification process involves using molecularmodels in which a spherical probe of radius 1.4 A, which is approximateto a water molecule, is used to track the surface of the molecule. Acavity, can accommodate a water molecule, i.e. the probe that is theequivalent size of a water molecule can fit within the cavity.Accordingly, a cavity has dimensions and a volume which can be measured.

As used herein, the term “functionally critical site” is meant to referto a site or region or location or secondary structural element on atarget protein that is involved in either altering or mediating afunction, the modulation of which is desirable. According to someembodiments of the invention, the function can be processing of amodifier that is an enzyme substrate by a target protein that is anenzyme, the functionally critical site is a target protein:modifierinteraction site that is a catalytic site, and the desirable modulationis the inhibition of substrate processing by the enzyme. According tosome embodiments of the invention, the function can be binding of themodifier to the target protein, the functionally critical site is atarget protein:modifier interaction site that is a binding site, and thedesirable modulation is the inhibition of target protein-modifierbinding. Other examples of functionally critical sites include surfacesof a target protein which interface with an oligomer and loops thatstabilize oligomers.

As used herein, the term “proximal” is used interchangeably with theterm “adjacent to” and is meant to refer to the distinct locations ofthe cavity and a functionally critical site which is at a measurabledistance. According to the invention, the cavity is at a distinctlocation from the functionally critical site. The two locations aredistinct from each other so that the modification of the functionallycritical site that occurs when a functional group of a compound occupiesthe cavity is allosteric modification. A cavity is proximal to afunctionally critical site if the functionally critical site can bealtered by molecular interactions between the target protein and atleast a functional group of a compound which can be accommodated by thecavity. In preferred embodiments, a cavity that is proximal to afunctionally critical site is generally within about 15-20 Angstroms tothe functionally critical site.

As used herein, the term “modulate” is meant to refer to an effect uponintermolecular interactions may be caused by compounds according to theinvention which allosterically modify molecular surfaces involved insuch intermolecular interactions. Such compounds are referred to hereinas “modulators”. In some embodiments, the effect caused by a modulatormay be the inhibition of intermolecular interactions, in which case themodulator is an “inhibitor”. In some embodiments, the effect caused by amodulator may be the enhancement of intermolecular interactions, inwhich case the modulator is an “enhancer”.

According to the invention, modulators, such as inhibitors or enhancers,of intermolecular interactions may be identified or designed toallosterically modify molecular surfaces involved in such intermolecularinteractions. Thus, any intermolecular interactions between a targetprotein that has a cavity proximal to a functionally critical site suchas a binding site or catalytic site, and a second molecule, a modifierwhich may or may not be a protein can be affected using compoundsidentified according to the invention.

The invention comprises a series of steps including: 1) identifying acavity proximal to a functional critical site; 2) determining physicalparameters of the cavity, 3) identifying functional groups which can beaccommodated by the cavity; and 4) testing compounds which comprise suchfunctional groups in an in vitro assay to determine whether suchcompounds are active.

According to the invention, the target protein must interact with amodifier and have a cavity proximal to a functional site. By identifyingfunctionally critical sites and cavities of a target protein ormodifier, which can be done routinely, it has been discovered that suchcavities, if proximal to the functionally critical site, can be targetsfor compounds that can modulate the activity of the target protein withrespect to its interaction with modifiers. Since the interaction withmodifiers is necessary for a specific biological function attributed tothe target protein, inhibition of target protein:modifier interactioninhibits the biological function associated with such interaction.Likewise, the enhancement of target protein:modifier interaction mayenhance the biological function associated with such interaction.

The means to identify functionally critical sites on a target proteinare numerous, varied and well known. For example, the identification ofactive or catalytic sites of enzymes, and the binding sites of receptorsor ligands are well known. The functionally critical site of a targetprotein may be identified several different ways including, but notlimited to: by identification of β-factors on the target proteinstructure as imaged using crystal or nuclear magnetic resonance (NMR)images either by thermal β-factors on the atoms of target protein fromcrystal structure or flexible loops inferred from NMR signals, ormicrocalorimetric analysis of complex or mutation analysis of molecule;by protein, peptide or peptidomimetic mapping of the target proteinincluding immunomapping; and by identifying CDRs on the target proteinstructure. β-factors are parameters that define flexibility such asthermal parameters from crystallographic studies. Thermal β-factors areparameters that reflect the disordered (flexible) nature of atoms in the3D structure determined by X-ray diffraction. When the structures aredetermined by X-ray diffraction, the data needed to determine β-factorsare measured as diffracted intensities. Fourier transform analysis ofthese data reveal the β-factors associated with the atoms in themolecule and β-factors always determined as a part of the crystalstructure analysis. These β-factors reflect the disorder or flexibilityof the atoms in the molecule. Calorimetric values from thermodynamicstudies can also be used to identify functionally critical site of atarget protein. An algorithm has been described which is also useful toidentify mobile regions. This algorithm and its use are described inDaquino, J. A. et al., 1996, Proteins, 25:143-156; Gomez, J. et al.,1995, Journal of Molecular Biology, 252:337-350; Hilser, V. J. et al.,1996, J. Mol. Biol., 262:756-772; and Xie, D. et al., Protein Science,3:2175-2184; which are each incorporated herein by reference.

The cavity of the target protein may be identified by any of severalwell known techniques including, but not limited to, crystal structureanalysis, NMR and computer models. The cavity size must be able toaccommodate at least one water molecule. The techniques for identifyingcavities on the surface of proteins are well known and described forexample in “Protein Engineering”, Edited by Dale L. Oxender, C. FredFox, Liss Co., New York (1987) (for crystallography & NMR) and“Guidebook on Molecular Modeling in Drug Design”, Edited by N. ClaudeCohen, Academic Press, 1996 San Diego, Calif. (1996) (for computermodeling), which are each incorporated herein by reference. To determinewhether a surface can accommodate water, using the computer model of theprotein, the surface is probed with small sphere of radius 1.4 A, a sizesimilar to that of a water molecule. The atoms touched by the probesphere are marked as surface atoms. Mapping the surface atoms as acontinuous surface defines the geometry of the surface. The geometrythen allows one to classify cavities. To be proximal the cavity must notbe at the same location as the functionally critical site.

Once a cavity that is proximal to the functionally critical site isidentified, certain physical parameters are ascertained. Such parametersinclude at least one and preferably more than one of the following: thevolume and dimensions of the cavity are measured or otherwisecalculated; the electrostatic properties of the cavity and/or thechemical properties, i.e. hydrophobicity/hydrophilicity, of the cavitymay be mapped. The interior of the cavity is thus defined by the volumeand dimensions of the interior of the cavity and/or the map ofelectrostatic properties within the interior of the cavity and/or themap of chemical properties within the interior of the cavity.

In some embodiments, the volume and dimensions of the interior of thecavity can be determined by rolling a probe radius of 1.4 A (equivalentof one water molecule) to generate a surface. The accessible surface isthen calculated using among many other programs freely available theprogram MS (Michael S. Connolly). MS is available from QCPE (QCPE,Creative Arts Bldg., 181, Indiana University, Bloomington, Ind. 47405)for and also packaged in several graphic software such as INSIGHT andQUANTA (both available from Molecular Simulations, Inc. San Diego,Calif.). In addition, the program described in Kleywegt, G. J. et al.,1994, Acta, D50:178-185, which is incorporated herein by reference, canalso be used to detect, measure and characterize cavities.

The electrostatic properties and chemical properties, i.e.hydrophobicity/hydrophilicity, of the cavity can be mapped. The residuesin the binding region are analyzed for site-points (atoms that arecapable of forming hydrogen bonds, hydrophobic interactions) using theprogram GENSITES. Other equivalent programs such SPHGEN which is part ofDOCK can also be used. The DOCK program is available from Prof. Kuntzlaboratory, University of California at San Francisco, San FranciscoCalif. The program identifies possible locations based upon differencesin surface accessibility of different sized spheres rolling over themolecular surface of the target protein. A three dimensional map of theinterior of the cavity is generated which corresponds to the dimensions,charge and chemical properties of the interior surfaces.

Once physical parameters of the cavity are ascertained, functionalgroups are identified which can be accommodated by the cavity. Suchfunctional groups must be of an appropriate size such that they can fitwithin the interior of the cavity. Additionally, functional groups mustbe electrostatically and chemically compatible with interior of thecavity. That is, the functional group must have electrostatic propertiesand chemical properties which would result in forces that attract thefunctional group to the interior of the cavity rather than repellingforces which would inhibit or prevent the functional group fromoccupying the interior of the cavity. Using the site points developed inthe cavity, possible molecular fragments are identified using theprogram LUDI. LUDI is part of INSIGHT which is available from MolecularSimulations, Inc. Another program from QUANTA called CAVEAT, alsoavailable from Molecular Simulations, Inc., can be used to identifyfunctional groups which can be accommodated by the cavity.

In some preferred embodiments, shape complementarity is used as initialscreen for detecting fragments with different moieties. The molecularmodeling approach assumes that the site is relatively rigid and that theintramolecular energy change upon target protein/modulator binding issmall compared to the interaction energy between target protein/modifierconformations. Therefore, the binding mode specifies which molecularpoint (expressible as Cartesian coordinates) on the modulator should bebound to which site point (also Cartesian coordinates) at the bindingsite. The fitting procedure is tantamount to identifying common surfacefeatures with subsequent docking of complementary surfaces.

Docking between two complementary surfaces can be an exhaustiveprocedure even with known topography. First, a probe sphere is rolled onthe binding surface as the locus of the possible positions which can beoccupied by the atoms of the binding molecule. This continuum of locican be reduced to a set of discrete points localized at each residue andassigned a type. An additional type assignment for each site point isgiven depending on the relative geometric description of this residuewith its three closest neighbors. These points define regions forfitting fragments identified in the LUDI data base. Complexes aresubjected to energy minimization and molecular dynamics calculations tooptimize the relative orientations and to monitor conformational changesin the target protein that are induced upon complex formation. Thisprocedure is done using AUTODOCK (Goodsell, et al. 1996 which isincorporated herein by reference) and LIGIN (Sobolev, et al. 1996 whichis incorporated herein by reference) or any other equivalent programsthat use docking algorithms. These two methods allow exploration of bothconformational flexibility and possible chemical modification forenhanced binding properties. This approach provides an estimate of thesize of the molecule that can bind and identify possible functionalgroups that can interact with neighboring residues and provides a way todevelop novel molecular structures based on the distribution of sitepoints.

Novel molecular compounds based on site points may encounter difficultyin synthesis and suitability for biological assays. To overcome thisobstacle, large three-dimensional chemical structure databases (MDLCorp., San Leandro, Calif.) are searched to identify compounds (Good, A.C. et al., 1995, J. Comput. Aided Mol. Des., 9:1-12; Kuntz, I. D., 1992,Science, 257:1078-1082; and Li, S. et al., 1997, Proc. Natl. Acad. Sci.U.S.A., 94:73-78; which are each incorporated herein by reference). Theadvantage of using the chemical database is two fold: (1) it offers aunique opportunity to search for novel molecules to small fragments thatcan be easily incorporated in a larger compound and (2) selection ofchemical compounds is facilitated from the knowledge of theiravailability, synthetic pathway, toxicity, and solubility etc.Currently, the three-dimensional structure chemical database containsabout 250,000 small molecules. Therapeutically useful compounds can beidentified in the chemical databases using the DOCK (Good, A. C. et al.,1995, J. Comput. Aided Mol. Des., 9:1-12 and Goodsell, D. S. et al.,1996, J. Mol. Recogn., 9:1-5, which are both incorporated herein byreference) algorithm. The cavity is explored with each small moleculefrom the database for maximal interaction such as hydrophobic, hydrogenbonds and complement electrostatic properties by conformational search.Based on the binding energy, the molecules are ranked and, for example,the top 200 compounds are selected. In addition, molecules similar tothe one constructed from de novo ligand design can be identified in thedatabases using a three-dimensionally constrained fragment search. Theshort listed molecules obtained both by database search (DOCK) andfragment search are used to create a small chemical database libraryusing MDL's project library software. Quantitative Structure ActivityRelation (QSAR) analysis in medicinal chemistry and pharmacology hasproven useful in making predictions for molecules that are chemicallysimilar to those of the original data set. Distance geometry directedQSAR allows for testing of a much wider class of compounds due to itsindependence from physico/chemical parameters. The molecules in thelibrary are compared for a common motif and analysis similar to 3D-QSARare carried out using ASP and TSAR (Oxford Molecular, Oxford, England)sequentially to find the suitable functional groups for maximal bindingenergy.

Following identification, compounds selected by one of the variousapproaches or combinations thereof are evaluated for biological activityin an in vitro assay to determine whether they modulate target-modifierinteractions. Biological assays are utilized for which intermolecularinteractions are known to result in a detectable signal or phenotype orfor which it is known that inhibition of intermolecular interactionsresult in a detectable signal or phenotype. Using such assays,comparative assays are performed in the presence or absence of theidentified compounds to confirm biological activity of the compound.

Pharmaceutical compositions according to the invention includecomponents identified by the methods of the invention which furthercomprise a pharmaceutically acceptable carriers or vehicles, such as,for example, saline. Any medium may be used which allows for successfuldelivery of the compound. One skilled in the art would readilycomprehend the multitude of pharmaceutically acceptable media that maybe used in the present invention. The term “pharmaceutical” is wellknown and widely understood by those skilled in the art. As used herein,the terms “pharmaceutical compositions” and “injectable pharmaceuticalcompositions” are meant to have their ordinary meaning as understood bythose skilled in the art. Pharmaceutical compositions, such asinjectable pharmaceutical compositions, are required to meet specificstandards regarding sterility, pyrogens, particulate matter as well asisotonicity and pH, i.e. inter alia sterile, pyrogen-free and free ofparticulate matter.

Pharmaceutical compositions may be formulated by one having ordinaryskill in the art with compositions selected depending upon the chosenmode of administration. Suitable pharmaceutical carriers are describedin Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, Ed.,Mack Publishing Company, Easton, Pa., a standard reference text in thisfield, which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. Pharmaceuticalcompositions may be administered parenterally, i.e., intratumor,intravenous, subcutaneous, intramuscular. Intravenous and intratumoradministration are preferred routes. For example, in cases whereintramuscular injection is the chosen mode of administration, anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.

Dosage varies depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired.

EXAMPLES Example 1

In one embodiment of the invention, CIAM technology is used to identifycompounds that inhibit interactions between tumor necrosis factor (TNF)receptor and TNFα.

Tumor necrosis factor receptor is one of the first receptors to bestudied at the atomic detail both as a complex and uncomplexedpolypeptide. The crystal structure of the TNF receptor both in complexedand uncomplexed forms provides a general understanding by which thesereceptors bind to their ligands (Banner, D. et al., 1993, Cell,73:431-45; Eck, M. J., et al. 1989, J. Biol. Chem., 264:17595-605; andEck, M. J., et al. 1992, J. Biol. Chem., 267:2119-22; which are eachincorporated herein by reference) and associated ligand inducedconformational changes. The cystine knot in the TNF receptor familyconsists of 42 amino acid residues with 6 cystine residues forming threeinter chain disulfide bond to create the structural motif. The threedimensional structure reveals the cystine-knots repeats about 30 Å inlength are arranged in a head-to-tail fashion exposing the loops on oneside of the receptor. These loops are either involved in oligomerizationor ligand binding. Uncomplexed TNF receptors are observed as dimers. Inthe dimeric form, the first and last cystine domains involved dimericcontacts. The membrane proximal domain is disordered perhaps due to thelack of the transmembrane that normally holds this domain in a stablestate. Crystal structure analysis of TNF receptor and TNFβ complex showsthat there are three distinct binding sites, referred to as “WP5”, “WP8”and “WP9”.

To understand, the most energetically relevant binding sites, peptideswere used as probes and several cyclic peptides were developed forspecies.

Peptidomimetics were developed and tested from all three surface loopsof the TNF receptor: loop (56-73) of domain 1; loop (76-83) of domain 2and loop (107-114) of domain 3 (FIG. 1A). The peptidomimetics aredescribed in detail in Takasai, et al. 1997 Nature Biotechnology15:1266-1270, which is incorporated herein by reference. Thepeptidomimetic engineered from the third domain (WP9QY) inhibited TNFαbinding (IC₅₀=75 mM) to its receptor. Also, the peptidomimetic protectedcells against TNFα induced cell death when apoptosis was induced with 7pg of TNFα suggesting that the peptide specifically bind to TNFα. Thepeptidomimetic (WP9QY) is one of the first peptides to show anti-TNFαactivity (FIG. 1B).

Based on the effect of the small loop in the third domain of TNFreceptor identified by peptidomimetic analysis, a large cleft wasidentified that could be utilized for docking an allosteric inhibitor.The cavity is shallow: 8 Å deep, 17.6 Å long and 12.4 Å wide (FIG. 2A).The walls of the cavity are formed by residues involved in binding TNFα.The large cleft close to one of the binding sites (WP9) was used toperturb these loops by an allosteric effect, using a small moleculedesigned from the above procedures (DesJarlais, R. L. et al., 1986, J.Med. Chem., 29:2149-2153; DesJarlais, R. L. et al., 1988, J. Med. Chem.,3:722-729; Good, A. C. et al., 1995, J. Comput. Aided Mol. Des., 9:1-12;Gschwend, D. A. et al., 1996, J. Mol. Recogn., 9:175-186; Shoicet, B. K.et al., 1991, J. Mol. Biol., 221:327-346; Strynadka, N. C. et al., 1996,Nat. Struct. Biol., 3:233-239; and Strynadka, N. C. et al., 1996, Nat.Struct. Biol., 3:290-297; which are each incorporated herein byreference). About 232 structurally suitable molecules were selected froman initial screening of small chemical database built using MDL (MDLcorporation, San Leandro, Calif.) structural chemical databasecontaining about 10000 structures. Further analysis revealed that notall of them are conducive for biological experiments based on solubilityand toxicity. For the purpose of testing, four compounds were tested forbiological activity, but not for specificity and kinetics. The compoundswere tested for apoptosis using a standard MTT assay (Hansen, M. B. etal., 1989, J. Immunol. Meth., 119:203-210, which is incorporated hereinby reference). One compound, S7 with binding energy of −40 Kcal/mol(FIG. 2B) showed activity (FIG. 2C) in the MTT assay. These resultsindicate that small molecules can be developed as pseudo-allostericinhibitors.

The present invention provides a novel strategy to modify theconformation of specific loops in an approach referred to as cavityinduced allosteric modification (CLAM) between the target protein, TNFreceptor, and the modifier, TNF. This strategy uses the known crystalstructure of the TNF receptor. The surface of the target protein wasgenerated by rolling a probe radius of 1.4 Å (equivalent of watermolecule). The accessible surface was calculated using program MS(Connolly, M. L. et al., 1993, J. Mol. Graph, 11:139-141 and Langridge,R. et al., 1981, Science, 211:661-666, which are both incorporatedherein by reference). The residues in the binding region are analyzedfor site-points (atoms that are capable of forming hydrogen bonds,hydrophobic interactions) using the program GENSITES. which identifiespossible locations based upon differences in surface accessibility ofdifferent sized spheres rolling over the molecular surface of the targetprotein. Using the site points developed in the cavity, possiblemolecular fragments were identified using the program LUDI (Bohm, L. W.et al., J. Mol. Recogn., 6:131-137 which is incorporated herein byreference). Shape complementarity was used as initial screen fordetecting fragments with different moieties. The molecular modelingapproach assumes that the site is relatively rigid and that theintramolecular energy change upon ligand/inhibitor binding is smallcompared to the interaction energy between receptor/proteinconformations. Therefore, the binding mode specifies which molecularpoint (expressible as Cartesian coordinates) on the inhibitor should bebound to which site point (also Cartesian coordinates) at the bindingsite. The fitting procedure is tantamount to identifying common surfacefeatures with subsequent docking of complementary surfaces.

Docking between two complementary surfaces can be an exhaustiveprocedure even with known topography. However, one can reduce thedimensionality of the problem by computing site points on the bindingsurface. First, a probe sphere is rolled on the binding surface as thelocus of the possible positions which can be occupied by the atoms ofthe binding molecule. This continuum of loci can be reduced to a set ofdiscrete points localized at each residue and assigned a type. Anadditional type assignment for each site point is given depending on therelative geometric description of this residue with its three closestneighbors. These points define regions for fitting fragments identifiedin the LUDI data base. Complexes are subjected to energy minimizationand molecular dynamics calculations to optimize the relativeorientations and to monitor conformational changes in the ligand thatare induced upon complex formation. This procedure is done usingAUTODOCK (Goodsell, D. S. et al., 1996, J. Mol. Recogn., 9:1-5 which isincorporated herein by reference) and LIGIN (Sobolev, et al. 1996Proteins, 25:120-129 which is incorporated herein by reference). Thesetwo methods allow exploration of both conformational flexibility andpossible chemical modification for enhanced binding properties. Thisapproach provides an estimate of the size of the molecule that can bind,identifies possible functional groups that can interact with neighboringresidues, and provides a way to develop novel molecular structures basedon the distribution of site points. Often, novel molecular compoundsbased on site points encounter difficulty in synthesis and suitabilityfor biological assays.

To overcome this obstacle, large three-dimensional chemical structuredatabases (MDL Corp., San Leandro, Calif.) are searched to identifycompounds (Good, A. C. et al., 1995, J. Comput. Aided Mol. Des., 9:1-12;Kuntz, I. D., 1992, Science, 257:1078-1082; and Li, S. et al., 1997,Proc. Natl. Acad. Sci. U.S.A., 94:73-78; which are each incorporatedherein by reference). The advantage of using the chemical database istwo fold: (1) it offers a unique opportunity to search for novelmolecules to small fragments that can be easily incorporated in a largercompound and (2) selection of chemical compounds is facilitated from theknowledge of their availability, synthetic pathway, toxicity, andsolubility etc. Currently, the three-dimensional structure chemicaldatabase contains about 250,000 small molecules. Therapeutically usefulcompounds can be identified in the chemical databases using the DOCK(Good, A. C. et al., 1995, J. Comput. Aided Mol. Des., 9:1-12 andGoodsell, D. S. et al., 1996, J. Mol. Recogn., 9:1-5, which are bothincorporated herein by reference) algorithm. The cavity is explored witheach small molecule from the database for maximal interaction such ashydrophobic, hydrogen bonds and complement electrostatic properties byconformational search. Based on the binding energy, the molecules areranked and the top 200 compounds are selected. In addition, moleculessimilar to the one constructed from de novo ligand design can beidentified in the databases using three-dimensionally constrainedfragment search. The short listed molecules obtained both by databasesearch (DOCK) and fragment search are used to create a small chemicaldatabase library using MDL's project library software. QuantitativeStructure Activity Relation (QSAR) analysis in medicinal chemistry andpharmacology has proven useful in making predictions for molecules thatare chemically similar to those of the original data set. Distancegeometry directed QSAR allows for testing of a much wider class ofcompounds due to its independence from physico/chemical parameters. Themolecules in the library are compared for a common motif and analysissimilar to 3D-QSAR are carried out using ASP and TSAR (Oxford Molecular,Oxford, England) sequentially to find the suitable functional groups formaximal binding energy. Finally, compounds selected from differentapproaches are evaluated using biological activities.

Cytotoxicity Assay

The murine fibroblast cell line, L929 is maintained in Dulbecco'smodified Eagle's medium supplemented with 10% FCS, and the medium isreplaced with serum free AIM-V medium (GIBCO BRL) right before seedingof the cells for an assay. L929 cells are seeded onto 96-well microtiterplates (2×10⁴ cells/well), and incubated for 20 hr at 37° C. under 5%CO₂ in air. After preincubation with actinomycin D (ACT-D) for 2 hr at afinal concentration of 1 mg/ml, TNFα (7 pg)/inhibitor solution (100-80ml), preincubated in PBS for 1 hr at 37° C., is added to the wells. Thecells are incubated with TNFa finally adjusted to 50 pg/ml for 7 hr at37° C. under 5% CO₂, and stained with MTT (Sigma). Briefly, 10 ml of the10 mg/ml solution of MTT is added to each well, and after 2 hrincubation at 37° C., the formazan formed is colored by overnightincubation at 37° C. with 100 ml of extraction buffer (20% SDS in 50%DMF, pH 4.7). Finally the optical density of colored formazan ismeasured at 600 nm.

Competitive Radioreceptor Assay

TNF-receptor chimeric protein (100 ng/ml) diluted in PBS (100 ml) isimmobilized onto MicroTest III flexible assay plate (Becton Dickinson,San Jose, Calif.) by an incubation at 4° C. overnight. After blockingwith PBS containing 1% bovine serum albumin (BSA) for 2 hr at roomtemperature and subsequent washing with PBS containing 0.1% Tween 20(PBS-Tw), ¹²⁵I-TNFα (1 ng)/inhibitor solution (100 ml) preincubated inPBS for 1 hr at 37° C. are added onto the TNF-receptor coated wells.After 2 hr incubation at 37° C., the plate is washed with PBS-Tw, andbound radioactivity is measured in Cobra gamma counter (PackardInstruments, Meriden, Conn.).

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula I which is set forth in thesection below entitled Formulae. In compound of Formula I, R₁ and R₂are, independently, selected from the group consisting of —H, —OCH₃,—CH₂CH₃, -t-butyl, 3-carboxy-4-chlorophenylamino, —N—(CH₂CH₂OH)₂, and—O(O)C-Ph. R₃ is selected from the group consisting of —H, ethyl, —OCH₃,—Cl, Br, F, 3-carboxy-4-chlorophenylamino, —N—(CH₂CH₂OH)₂, -t-butyl, and—OC(O)-Ph, and is not limited to attachment at any certain position onthe phenyl ring to which it is attached. Preferably, R₃ is attached ateither the 1 or 4 position of the phenyl ring. R₄ is selected from thegroup consisting of —Br, —Cl, and —F.

In some preferred compounds of Formula I

-   -   R₁, R₂, and R₃ are —OCH₃, R₃ is attached at the 4 position, R₄        is —Cl;    -   R₁ and R₂ are methyl, R₃ is ethyl, attached at the 4 position,        R₄ is —Cl    -   R₁ and R₂ are —OCH₃, R₃ is —Cl, attached at the 2 position, R₄        is —Cl;    -   R₁ and R₂ are —OCH₃ and R₃ is H, R₄ is —Cl;    -   R₁ is H, R₂ and R₃ are 3-carboxy-4-chlorophenylamino, and R₃ is        attached at the 4 position, R₄ is —Cl;    -   R₁ and R₂ are —N(CH₂CH₂OH)₂, R₃ is Cl, attached at the 4        position, R₄ is —Cl;    -   R₁, R₂, and R₃ are t-butyl, R₃ is attached at the 4 position, R₄        is —Cl;    -   R₁ is —OCH₃, R₂ and R₃ are H, R₄ is Cl; or    -   R₁, R₂, and R₃ are benzoate, R₃ is attached at the 4 position,        R₄ is —Br.

Some preferred compounds of Formula I have the structures I-A, I-B, I-C,I-D, I-E, I-F, I-G, I-H or I-I which are set forth below in the sectionentitled Formulae.

These compounds are available from the following suppliers:

Compound Catalog Number Supplier I-A F36,700-1 Aldrich, Milwaukee, WII-B S11,245-3 Aldrich, Milwaukee, WI I-C 00569 Ryan Scientific, Isle ofPalms, S.C. I-D F10,001-3 Aldrich, Milwaukee, WI I-E 00129 George UHE,Paramus, NJ I-F F37,166-1 Aldrich, Milwaukee, WI I-G S-11,239-9 Aldrich,Milwaukee, WI I-H F-27,721-5 Aldrich, Milwaukee, WI I-I F12,920-8Aldrich, Milwaukee, WI

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula H which is set forth belowin the section entitled Formulae. In compounds having Formula II, R₁ isselected from the group consisting of -diphenylchloro methyl,-di(4-chlorophenyl)chloro methyl, and 4-(diphenylchloromethyl)phenyl;and R₂, R₃, R₄ are independently selected from the group consisting of—Br, —Cl, and —F, and are preferably —Cl.

Preferred compounds of Formula II have the structures II-A, II-B, II-Cand II-D which are set forth below in the section entitled Formulae.These compounds are available from the following suppliers:

Compound Catalog Number Supplier II-A S5,479-9 Aldrich, Milwaukee, WIII-B S5,755-0 Aldrich, Milwaukee, WI II-C S5,740-2 Aldrich, Milwaukee,WI II-D S5,751-8 Aldrich, Milwaukee, WI

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula III which is set forth inthe section below entitled Formulae. In compound of Formula III, R₁ is Hor diethylamino; R₂ is —O— or —N(C₆H₆)—, and R₃ is —Br, Cl, or F.

Preferred compounds of Formula III have the structure III-A and III-Bwhich are set forth below in the section entitled Formulae.

These compounds are available from the following suppliers:

Compound Catalog Number Supplier III-A F21,855-5 Aldrich, Milwaukee, WIIII-B C-390 Biosynth, Naperville, IL

Example 2

Pharmaceutical compositions are prepared using compounds of Formulas I,II and III which are commercially available from chemical suppliers suchas Sigma, Aldrich, ICN, Ryan Scientific, George Uhe (Paramus, N.J.), andBiosynth (Naperville, Ill.). The pharmaceutical compositions are usefulto treat individuals suffering from TNF-mediated diseases, disorders andconditions. Examples of TNF-mediated diseases, disorders and conditionsinclude, for example, inflammatory diseases and autoimmune diseases suchas rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren'ssyndrome, sarcoidosis, insulin dependent diabetes mellitus (IDDM),autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis,scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis,Wegener's granulomatosis, Crohn's disease, ulcerative colitis, Lupus(SLE), Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliarysclerosis and pernicious anemia. According to the present invention,individuals suffering from such diseases, disorders and conditions maybe treated by administering to them a therapeutically effective amountof a pharmaceutical composition that comprises a compound having FormulaI, II and III.

The method may include administration of compounds to mammals,preferably humans, in therapeutically effective amounts which areeffective to inhibit TNF-mediated diseases. The dosage administered inany particular instance will depend upon factors such as thepharmacodynamic characteristics of the compound, its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency oftreatment, and the effect desired.

It is contemplated that the daily dosage of a compound used in themethod that is the invention will be in the range of from about 1 μg toabout 10 grams per day. In some preferred embodiments, the daily dosagecompound will be in the range of from about 10 mg to about 1 gram perday. In some preferred embodiments, the daily dosage compound will be inthe range of from about 100 mg to about 500 mg per day. It iscontemplated that the daily dosage of a compound used in the method thatis the invention will be in the range of from about 1 μg to about 100 mgper kg of body weight, in some embodiments, from about 1 μg to about 40mg per kg body weight; in some embodiments from about 10 μg to about 20mg per kg per day, and in some embodiments 10 μg to about 1 mg per kgper day.

Pharmaceutical compositions may be administered in a single dosage,divided dosages or in sustained release. In some preferred embodiments,the compound will be administered in multiple doses per day. In somepreferred embodiments, the compound will be administered in 3-4 dosesper day.

Persons of ordinary skill will be able to determine dosage forms andamounts with only routine experimentation based upon the considerationsof this invention.

The method of administering compounds include administration as apharmaceutical composition orally in solid dosage forms, such ascapsules, tablets, and powders, or in liquid dosage forms, such aselixirs, syrups, and suspensions. The compounds may also be administeredparenterally in sterile liquid dosage forms or topically in a carrier.The compounds may be formulated into dosage forms according to standardpractices in the field of pharmaceutical preparations. See Remington'sPharmaceutical Sciences, 18^(th) Edition, A.R. Gennaro, Ed., MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

Compounds may be mixed with powdered carriers, such as lactose, sucrose,mannitol, starch, cellulose derivatives, magnesium stearate, and stearicacid for insertion into gelatin capsules, or for forming into tablets.Both tablets and capsules may be manufactured as sustained releaseproducts for continuous release of medication over a period of hours.

Liquid dosage forms for oral administration may contain coloring andflavoring to increase patient acceptance, in addition to apharmaceutically acceptable diluent such as water, buffer or salinesolution.

For parenteral administration, a compound may be mixed with a suitablecarrier or diluent such as water, a oil, saline solution, aqueousdextrose (glucose), and related sugar solutions, and glycols such aspropylene glycol or polyethylene glycols. Solutions for parenteraladministration contain preferably a water soluble salt of the compound.Stabilizing agents, antioxidizing agents and preservatives may also beadded. Suitable antioxidizing agents include sodium bisulfite, sodiumsulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.Suitable preservatives include benzalkonium chloride, methyl- orpropyl-paraben, and chlorbutanol.

Example 3

In one embodiment of the invention, CIAM technology is used to identifycompounds that inhibit interactions between CD4, and MHC/antigen/TCRcomplexes.

By inhibiting CD4, the T cell activation associated with MHC/antigen/TCRcomplexes can be reduced and immune responses suppressed accordingly.The crystal structure of the CD4 complex shows distinct binding sites.

Peptidomimetics were developed and tested from surface loops of CD4. Apeptidomimetic engineered from a CD4 domain inhibited T cell activationassociated with MHC/antigen/TCR complexes.

Using the location of the functionally active amino acid sequenceidentified using the peptidomimetic as the location of a functionallycritical active site, the surface of the CD4 molecule was reviewed and alarge cleft was identified that could be utilized for docking apseudo-allosteric inhibitor. The interior of the cavity was mapped and achemical database was searched. A compound with binding energy of −34Kcal/mol and having Formula IV was identified. This compound which iscommercially available from Salor/Aldrich (Catalog #S69, 246-8) showedactivity in in vitro T cell activation assays.

Pharmaceutical compositions are prepared using the compound of FormulaIV, V or VI which are set forth in the section below entitled Formulae.The pharmaceutical compositions are useful to treat individualssuffering from CD4-mediated diseases, disorders and conditions. Examplesof CD4-mediated diseases, disorders and conditions include, for example,inflammatory diseases and autoimmune diseases such as rheumatoidarthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmunethyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma,polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener'sgranulomatosis, Crohn's disease, ulcerative colitis, Lupus (SLE),Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliarysclerosis and pernicious anemia. According to the present invention,individuals suffering from such diseases, disorders and conditions maybe treated by administering to them a therapeutically effective amountof a pharmaceutical composition that comprises a compound having eitherFormula IV, V or VI.

The method may include administration of compounds to mammals,preferably humans, in therapeutically effective amounts which areeffective to inhibit CD4-mediated diseases. The dosage administered inany particular instance will depend upon factors such as thepharmacodynamic characteristics of the compound, its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency oftreatment, and the effect desired.

It is contemplated that the daily dosage of a compound used in themethod that is the invention will be in the range of from about 1 μg toabout 10 grams per day. In some preferred embodiments, the daily dosagecompound will be in the range of from about 10 mg to about 1 gram perday. In some preferred embodiments, the daily dosage compound will be inthe range of from about 100 mg to about 500 mg per day. It iscontemplated that the daily dosage of a compound used in the method thatis the invention will be in the range of from about 1 μg to about 100 mgper kg of body weight, in some embodiments, from about 1 μg to about 40mg per kg body weight; in some embodiments from about 10 μg to about 20mg per kg per day, and in some embodiments 10 μg to about 1 mg per kgper day.

Pharmaceutical compositions may be administered in a single dosage,divided dosages or in sustained release. In some preferred embodiments,the compound will be administered in multiple doses per day. In somepreferred embodiments, the compound will be administered in 3-4 dosesper day.

Persons of ordinary skill will be able to determine dosage forms andamounts with only routine experimentation based upon the considerationsof this invention.

The method of administering compounds include administration as apharmaceutical composition orally in solid dosage forms, such ascapsules, tablets, and powders, or in liquid dosage forms, such aselixirs, syrups, and suspensions. The compounds may also be administeredparenterally in sterile liquid dosage forms or topically in a carrier.The compounds may be formulated into dosage forms according to standardpractices in the field of pharmaceutical preparations. See Remington'sPharmaceutical Sciences, 18th Edition, A.R. Gennaro, Ed., MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

Compounds may be mixed with powdered carriers, such as lactose, sucrose,mannitol, starch, cellulose derivatives, magnesium stearate, and stearicacid for insertion into gelatin capsules, or for forming into tablets.Both tablets and capsules may be manufactured as sustained releaseproducts for continuous release of medication over a period of hours.

Liquid dosage forms for oral administration may contain coloring andflavoring to increase patient acceptance, in addition to apharmaceutically acceptable diluent such as water, buffer or salinesolution.

For parenteral administration, a compound may be mixed with a suitablecarrier or diluent such as water, a oil, saline solution, aqueousdextrose (glucose), and related sugar solutions, and glycols such aspropylene glycol or polyethylene glycols. Solutions for parenteraladministration contain preferably a water soluble salt of the compound.Stabilizing agents, antioxidizing agents and preservatives may also beadded. Suitable antioxidizing agents include sodium bisulfite, sodiumsulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.Suitable preservatives include benzalkonium chloride, methyl- orpropyl-paraben, and chlorbutanol.

Example 4

In one embodiment of the invention, CIAM technology is used to identifycompounds that inhibit β-lactamase. Inhibition of the enzyme β-lactamaseis useful to render penicillin-resistant strains of bacteria,penicillin-sensitive. A cavity with the criteria described aboveproximal from the active site of β-lactamase was identified by β-factorsand thermodynamic analysis.

The surface of the β-lactamase molecule was reviewed and a proximalsuitable cleft was identified that could be utilized for docking anallosteric inhibitor. The cavity was mapped and a chemical database wassearched. A series of compounds were identified. These compounds areshown as Formulae VII-XIX in the section below entitled Formulae. Theyare commercially available from several suppliers including Aldrich(Milwaukee, Wis.; www.sigma-aldtich.com), Sigma (www.sigma-aldrich.com),Fluka (www.sigma-aldrich.com), ICN (www.icnpharm.com), Ryan Scientific(Isle of Palms, S.C.), SynTec (Germany) and Bayer (Leverkusen, Germany;www.bayer.com).

Pharmaceutical compositions are prepared using one of the compoundsselected from the group of Formula VII-XIX. The pharmaceuticalcompositions are useful to treat individuals suffering from bacterialinfectious, particularly those which are penicillin resistant. Accordingto the present invention, individuals suffering from such infections maybe treated by administering to them a therapeutically effective amountof a pharmaceutical composition that comprises a compound having FormulaVII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX incombination with penicillin-derived antibiotic.

The method may include administration of compounds to mammals,preferably humans, in therapeutically effective amounts which areeffective to inhibit β-lactamase in order to render penicillin-resistantstrains of bacteria penicillin-sensitive. The dosage administered in anyparticular instance will depend upon factors such as the pharmacodynamiccharacteristics of the compound, its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment, frequency of treatment, and the effectdesired.

It is contemplated that the daily dosage of the compound used in themethod that is the invention will be in the range of from about 1 μg toabout 10 grams per day. In some preferred embodiments, the daily dosagecompound will be in the range of from about 10 mg to about 1 gram perday. In some preferred embodiments, the daily dosage compound will be inthe range of from about 100 mg to about 500 mg per day. It iscontemplated that the daily dosage of a compound used in the method thatis the invention will be in the range of from about 1 μg to about 100 mgper kg of body weight, in some embodiments, from about 1 μg to about 40mg per kg body weight; in some embodiments from about 10 μg to about 20mg per kg per day, and in some embodiments 10 μg to about 1 mg per kgper day.

Pharmaceutical compositions may be administered in a single dosage,divided dosages or in sustained release. In some preferred embodiments,the compound will be administered in multiple doses per day. In somepreferred embodiments, the compound will be administered in 3-4 dosesper day.

Persons of ordinary skill will be able to determine dosage forms andamounts with only routine experimentation based upon the considerationsof this invention.

The method of administering compounds include administration as apharmaceutical composition orally in solid dosage forms, such ascapsules, tablets, and powders, or in liquid dosage forms, such aselixirs, syrups, and suspensions. The compounds may also be administeredparenterally in sterile liquid dosage forms or topically in a carrier.The compounds may be formulated into dosage forms according to standardpractices in the field of pharmaceutical preparations. See Remington'sPharmaceutical Sciences, 18^(th) Edition, A.R. Gennaro, Ed., MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

Compounds may be mixed with powdered carriers, such as lactose, sucrose,mannitol, starch, cellulose derivatives, magnesium stearate, and stearicacid for insertion into gelatin capsules, or for forming into tablets.Both tablets and capsules may be manufactured as sustained releaseproducts for continuous release of medication over a period of hours.

Liquid dosage forms for oral administration may contain coloring andflavoring to increase patient acceptance, in addition to apharmaceutically acceptable diluent such as water, buffer or salinesolution.

For parenteral administration, a compound may be mixed with a suitablecarrier or diluent such as water, a oil, saline solution, aqueousdextrose (glucose), and related sugar solutions, and glycols such aspropylene glycol or polyethylene glycols. Solutions for parenteraladministration contain preferably a water soluble salt of the compound.Stabilizing agents, antioxidizing agents and preservatives may also beadded. Suitable antioxidizing agents include sodium bisulfate, sodiumsulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.Suitable preservatives include benzalkonium chloride, methyl- orpropyl-paraben, and chlorbutanol.

In some embodiments, the pharmaceutical compositions of the presentinvention used in the methods of the present invention comprisecompounds having Formulae VII-XVI.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula VII.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula VIII.

Compounds according to Formula VIII may have at position R₁ a grouphaving Formula 8-1-1, 8-1-2, 8-1-3, 8-1-4, 8-1-5, 8-1-6, 8-1-7, 8-1-8,8-1-9 and 8-1-10 which are set forth below in the section entitledFormulae.

Compounds according to Formula VIII may have at position R₂ and R₂ are,independently, —H, —C₁, —C₂, —C₃ straight or branched, —C₄ straight orbranched, —C₅ straight or branched, C₆ straight or branched, —C₇straight or branched, and —C₈ straight or branched. R₂ and R₂ arepreferably identical to each other. R₂ and R₂ are preferably-H or the—C₈ branched 4-tert-octyl.

Some preferred compounds of Formula VIII include compounds wherein:

-   -   R₁ is 8-1-1, R₂ is —H, and R₃ is —H;    -   R₁ is 8-1-1, R₂ is 4-tert-octyl, and R₃ is 4-tert-octyl;    -   R₁ is 8-1-2, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-3, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-4, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-5, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-6, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-7, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-8, R₂ is —H and R₃ is —H;    -   R₁ is 8-1-9, R₂ is —H and R₃ is —H; and    -   R₁ is 8-1-10, R₂ is —H and R₃ is —H.

Some preferred compounds of Formula VIII include structure VIII-A,VIII-B, VIII-C, VIII-D, VIII-E, VIII-F, VIII-G, VIII-H, VIII I, VIII Jor which are set forth below in the section entitled Formulae.

Compund Source Catalog Number VIII-A. ALDRICH F28,168-9 VIII-B. ALDRICH25,762-1 VIII-C. SIGMA-ALDRICH S68,073-7 VIII-D. SIGMA-ALDRICH S15,490-3VIII-E. SIGMA-ALDRICH S15,495-4 VIII-F. SIGMA-ALDRICH S15,498-9 VIII-G.SIGMA-ALDRICH S15,504-0 VIII-H. SIGMA-ALDRICH S15,505-5 VIII-I.SIGMA-ALDRICH R17,712-1 VIII-J. SIGMA-ALDRICH R17,271-5 VIII-K.SIGMA-ALDRICH R17,703-2

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula IX.

Compounds according to Formula IX may have at position R₁, R₂, R₃ andR₄, independently, —H, —OC₆H₅Cl, —N(CH₃)₂, —OCH₃, —CH₃, —OH or -halogen,and if halogen, preferably —Cl. In some preferred embodiments, R₁ is—OCH₃, —OH or -halogen, and if halogen, preferably —Cl. In somepreferred embodiments R₂ is —H, —N(CH₃)₂, —OCH₃, —CH₃, or -halogen, andif halogen, preferably —Cl. In some preferred embodiments R₃ is —OCH₃,—CH₃, —OH or -halogen, and if halogen, preferably —Cl. In some preferredembodiments R₄ is —OC₆H₅Cl, —OCH₃, —CH₃ or —OH.

Some preferred compounds of Formula IX include compounds wherein:

-   -   R₁ is —Cl, R₂ is —H, R₃ is —Cl and R₄ is OC₆H₅Cl;    -   R₁ is —N(CH₃)₂, R₂ is —H, R₃ is —Cl and R₄ is —OCH₃;    -   R₁ is —OCH₃, R₂ is —H, R₃ is —OCH₃ and R₄ is —OCH₃;    -   R₁ is —Cl, R₂ is —H, R₃ is —Cl and R₄ is —OCH₃;    -   R₁ is —Cl, R₂ is —H, R₃ is —CH₃ and R₄ is —OCH₃;    -   R₁ is —OCH₃, R₂ is —Cl, R₃ is —H and R₄ is —OH;    -   R₁ is —OH, R₂ is —H, R₃ is —Cl and R₄ is —OCH₃;    -   R₁ is —OCH₃, R₂ is —H, R₃ is —CH₃ and R₄ is —OCH₃;    -   R₁ is —Cl, R₂ is —Cl, R₃ is —OCH₃ and R₄ is —OCH₃;    -   R₁ is —Cl, R₂ is —H, R₃ is —OCH₃ and R₄ is —OCH₃;    -   R₁ is —OCH₃, R₂ is —H, R₃ is —OCH₃ and R₄ is —OCH₃;    -   R₁ is —OCH₃, R₂ is —CH₃, R₃ is —Cl and R₄ is —H;    -   R₁ is —OH, R₂ is —H, R₃ is —Cl and R₄ is —CH₃; and    -   R₁ is —OCH₃, R₂ is —OCH₃, R₃ is —OH and R₄ is —H.

Some preferred compounds of Formula IX include structure IX-A, IX-B,IX-C, IX-D, IX-E, IX-F, IX-G, IX-H, IX-I, IX-J, IX-K, IX-L, IX-M or IX-Nwhich are set forth below in the section entitled Formulae.

Compund Source Catalog Number IX-A. SIGMA-ALDRICH S50,872-1 IX-B.SIGMA-ALDRICH S69,044-9 IX-C. SIGMA-ALDRICH S69,613-7 IX-D.SIGMA-ALDRICH S69-516-5 IX-E. SIGMA-ALDRICH S12,931-3 IX-F.SIGMA-ALDRICH S72,315-0 IX-G. SIGMA-ALDRICH S69,055-4 IX-H.SIGMA-ALDRICH S76,872-3 IX-I. SIGMA-ALDRICH S90,369-8 IX-J.SIGMA-ALDRICH S90,370-1 IX-K. SIGMA-ALDRICH S74,299-6 IX-L.SIGMA-ALDRICH S72,956-6 IX-M. SIGMA-ALDRICH S91,728-1 IX-N.SIGMA-ALDRICH S91,730-3

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula X.

Compounds according to Formula X may have at position R₁ a group havingFormula 10-1-1, 10-1-2 or 10-1-3 which are set forth below in thesection entitled Formulae.

Compounds according to Formula X may have at position R₂, R₃ and R₄ are,independently, —H, —NO₂, —NH₂ or —CH₃. R₂ is preferably —H or —NH₂. R₃is preferably —NO₂ or —NH₂. R₄ is preferably —H or —CH₃.

Some preferred compounds of Formula X include compounds wherein:

-   -   R₁ is 10-1-1, R₂ is —H, R₃ is —NO₂ and R₄ is —H;    -   R₁ is 10-1-2, R₂ is —H, R₃ is —NO₂ and R₄ is —H; and    -   R₁ is 10-1-3, R₂ is —NH₂, R₃ is —NH₂ and R₄ is —CH₃.

Some preferred compounds of Formula X include structure X-A, X-B or X-Cwhich are set forth below in the section entitled Formulae.

Compound Source Catalog Number X-A. RYAN SCIENTIFIC NRB01150 X-B. SIGMAS93,056-3 X-C. ALDRICH 21,222-9

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XI.

Compounds according to Formula XI may have at position R₁ and R₂,independently, a group having Formula 11-1/2-1, 11-1/2-2, 11-1/2-3,11-1/2-4, which are set forth below in the section entitled Formulae, or—H, —NO₂ or —OH

Compounds according to Formula XI may have at position R₃ either —H,—NH₂, —OH, or halogen, and when R₃ is halogen, it is preferably —Cl or—Br.

Compounds according to Formula XI may have at position R₃ either —H,—NH₂, —OH, or halogen, and when R₃ is halogen, it is preferably —Cl or—Br.

Compounds according to Formula XI may have at position R₄ either —H or—CH(CH₃)₃.

Some preferred compounds of Formula XI include compounds wherein:

-   -   R₁ is 11-1/2-1, R₂ is 11-1/2-1, R₃ is —H and R₄ is —H;    -   R₁ is 11-1/2-2, R₂ is 11-1/2-2, R₃ is —NH₂ and R₄ is —H;    -   R₁ is 11-1/2-3, R₂ is 11-1/2-3, R₃ is —Cl, and R₄ is —H;    -   R₁ is 11-1/2-4, R₂ is —H, R₃ is —NO₂ and R₄ is —H; and    -   R₁ is —NO₂, R₂ is —OH, R₃ is —OH and R₄ is —H.

Some preferred compounds of Formula XI include structure XI-A, XI-B,XI-C, XI-D or XI-E which are set forth below in the section entitledFormulae.

Compound Source Catalog Number XI-A. SIGMA-ALDRICH S18,982-0 XI-B.SIGMA-ALDRICH S18,611-2 XI-C. SIGMA S3,634-0 XI-D. SIGMA S86,927-9 XI-E.SIGMA S53,622-9

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XII.

Compounds according to Formula XII may have at position R₁ eitherN-pyridinium, 4-methyl-N-pyridinium, 4-dimethylamino-N-pyridinium,3-methyl-N-pyridinium, N-pyridinium, 2,6 dimethyl-N-pyridinium, 3,5dimethyl-N-pyridinium, 3-ethyl-N-pyridinium, 12-1-1, 12-1-2 which areset forth below in the section entitled Formulae, or4-ethyl-N-pyridinium, 4-benzyl-N-pyridinium, N-quinolinyl or CH₃.

Some preferred compounds of Formula XII include compounds wherein:

-   -   R₁ is N-pyridinium and R₂ is NO₂;    -   R₁ is 4-methyl-N-pyridinium and R₂ is NO₂;    -   R₁ is 4-dimethylamino-N-pyridinium and R₂ is NO₂;    -   R₁ is 3-methyl-N-pyridinium and R₂ is NO₂;    -   R₁ is N-pyridinium and R₂ is NO₂;    -   R₁ is 2,6 dimethyl-N-pyridinium and R₂ is NO₂;    -   R₁ is 3,5 dimethyl-N-pyridinium and R₂ is NO₂;    -   R₁ is 3-ethyl-N-pyridinium and R₂ is NO₂;    -   R₁ is 12-1-1 and R₂ is NO₂;    -   R₁ is 12-1-2 and R₂ is NO₂;    -   R₁ is 4-ethyl-N-pyridinium and R₂ is NO₂;    -   R₁ is 4-benzyl-N-pyridinium and R₂ is NO₂;    -   R₁ is N-quinolinyl and R₂ is NO₂; and    -   R₁ is CH₃ and R₂ is H.

Some preferred compounds of Formula XII include structure XII-A, XII-B,XII-C, XII-D, XII-E, XII-F, XII-G, XII-H, XII-I, XII-J, XII-K, XII-N,XII-M or XII-N, which are set forth below in the section entitledFormulae.

Compound Source Catalog Number XII-A. SIGMA S14,318-9 XII-B. SIGMAS96,676-2 XII-C. SIGMA S14,440-1 XII-D. SIGMA S96,664-9 XII-E. SIGMAS96,668-1 XII-F. SIGMA S96,670-3 XII.G. SIGMA S14,386-3 XII-H. SIGMAS96,674-6 XII-I. SIGMA S96,682-7 XII-J. SIGMA S96,677-0 XII-K. SIGMAS96,679-7 XII-L. SIGMA S96,685-1 XII-M. SIGMA S14,675-7 XII-N.SIGMA-ALDRICH S67,954-2

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XIII which is set forth inthe section below entitled Formulae. Compounds having Formula XIII areavailable from SIGMA-ALDRICH, Catalog number S42,591-5.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XIV.

Compounds according to Formula XIV may have at position R₁ either —OCH₃,—NO₂ or -halogen, and if -halogen, preferably —Cl.

Compounds according to Formula XIV may have at position R₂ either —H,—NO₂ or -halogen, and if -halogen, preferably —Cl.

Some preferred compounds of Formula XIV include compounds wherein:

-   -   R₁ is —Cl and R₂ is —Cl;    -   R₁ is —OCH₃ and R₂ is —H;    -   R₁ is —Cl and R₂ is —H; and    -   R₁ is —NO₂ and R₂ is NO₂.

Some preferred compounds of Formula XIV include structure)(IV-A, XIV-B,XIV-C or XIV-D which are set forth in the section below entitledFormulae.

Compound Source Catalog Number XIV-A. SIGMA S6,886-2 XIV-B. SIGMAS12,703-5 XIV-C. SIGMA S62,321-0 XIV-D. SIGMA S24,232-2

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XV.

Compounds according to Formula XV may have at position R₁ either —H,—NO₂, —FSO₂, —CH₃, —OCH₃, —SO₂CH₃, 15-1-1, 15-1-2 or 15-1-3 which areset forth in the section below entitled Formulae.

Compounds according to Formula XV may have at position R₂ either —H,—OH, or —NO₂.

Compounds according to Formula XV may have at position R₃ either —H or—OH.

Compounds according to Formula XV may have at position R₄ either —H,15-4-1, 15-4-2, 15-4-3, 15-4-4, 15-4-5, 15-4-6, 15-4-7, 15-4-8, 15-4-9,15-4-10, 15-4-11, 15-4-12 which are set forth in the section belowentitled Formulae or halogen, and when R₄ is halogen, it is preferably—Cl.

Some preferred compounds of Formula XV include compounds wherein:

-   -   R₁ is —NO₂, R₂ is —H, R₃ is —OH and R₄ is —Cl;    -   R₁ is —H, R₂ is —OH, R₃ is —H and R₄ is −15-4-1;    -   R₁ is —FSO₂, R₂ is —NO₂, R₃ is —OH and R₄ is —H;    -   R₁ is 15-1-1, R₂ is —NO₂, R₃ is —OH and R₄ is —H;    -   R₁ is —H, R₂ is —H, R₃ is —H and R₄ is 15-4-2;    -   R₁ is —H, R₂ is —H, R₃ is —OH and R₄ is 15-4-3;    -   R₁ is —H, R₂ is —H, R₃ is —OH and R₄ is 15-4-5;    -   R₁ is —H, R₂ is —OH, R₃ is —OCH₃ and R₄ is 15-4-6;    -   R₁ is —OCH₃, R₂ is —H, R₃ is —OH and R₄ is 15-4-7;    -   R₁ is —H, R₂ is —H, R₃ is —OH and R₄ is 15-4-8;    -   R₁ is —H, R₂ is —H, R₃ is —OH and R₄ is 15-4-9;    -   R₁ is —OCH₃, R₂ is —H, R₃ is —H and R₄ is 15-4-10;    -   R₁ is —FSO₂, R₂ is —H, R₃ is —OH and R₄ is 15-4-11;    -   R₁ is —H, R₂ is —H, R₃ is —H and R₄ is 15-4-12;    -   R₁ is —SO₂CH₃, R₂ is —H, R₃ is —OH and R₄ is —H;    -   R₁ is 15-1-2, R₂ is —H, R₃ is —OH and R₄ is —H; and    -   R₁ is 15-1-3, R₂ is —NO₂, R₃ is —OH and R₄ is —H.

Some preferred compounds of Formula XV include structure XV-A, XV-B,XV-C, XV-D, XV-E, XV-F, XV-G, XV-H, XV-I, XV-J, XV-K, XV-L, XV-M, XV-N,XV-O, XV-P, XV-Q or XV-R which are set forth in the section belowentitled Formulae.

Compound Source Catalog Number XV-A. SIGMA S72,767-9 XV-B. SIGMAS72,772-5 XV-C. SIGMA S73,689-9 XV-D. BAYER CORP. 25/08 XV-E. SIGMAS50,245-6 XV-F. SIGMA S65,507-4 XV-G. SIGMA S78,072-3 XV-H. SIGMAS79,426-0 XV-I. SIGMA S79,453-8 XV-J. SIGMA S80,012-0 XV-K. SIGMAS84,486-1 XV-L. RYAN NRB01429 XV-M. SIGMA S92,407-5 XV-N. SIGMAS72,781-4

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XVI.

Compounds according to Formula XVI may have at position R₁ either —H,—SO₃ or halogen, and when R₄ is halogen, it is preferably —Cl.

Compounds according to Formula XVI may have at position R₂ either —H or—CH₃.

Compounds according to Formula XVI may have at position R₃ either —H,—OCH₃, —NSO₂—NO₂, 16-3-1 which is set forth below in the sectionentitled Formulae, or halogen, and when R₃ is halogen, it is preferably—Fl.

Compounds according to Formula XVI may have at position R₄ either —H,—OCH₃, —SO₃ or halogen, and when R₄ is halogen, it is preferably —Cl.

Compounds according to Formula XVI may have at position R₅ either —H,—NO₂ or halogen, and when R₅ is halogen, it is preferably —Cl.

Compounds according to Formula XVI may have at position R₆ either —H,—C(CH₃)₂ or phenyl.

Some preferred compounds of Formula XVI include compounds wherein:

-   -   R₁ is —H, R₂ is —CH₃, R₃ is —OCH₃, R₄ is —OCH₃, R₅ is —H and R₄        is —H,    -   R₁ is —H, R₂ is —CH₃, R₃ is —NSO₂, R₄ is —H, R₅ is —H and R₆ is        —H,    -   R₁ is —H, R₂ is —CH₃, R₃ is —OCH₃, R₄ is —Cl, R₅ is —H and R₆ is        —H,    -   R₁ is —NSO₃, R₂ is —CH₃, R₃ is —OCH₃, R₄ is —SO₃, R₅ is —NO₂ and        R₆ is —H,    -   R₁ is —H, R₂ is —H, R₃ is —H, R₄ is —H, R₅ is —H and R₆ is —H,    -   R₁ is —H, R₂ is —H, R₃ is —Cl, R₄ is —Cl, R₅ is —H and R₆ is —H,    -   R₁ is —H, R₂ is —CH₃, R₃ is —F, R₄ is —H, R₅ is —H and R₆ is —H,    -   R₁ is —H, R₂ is —CH₃, R₃ is −16-3-1, R₄ is —H, R₅ is —H and R₆        is —H,    -   R₁ is —H, R₂ is —CH₃, R₃ is —H, R₄ is —H, R₅ is —H and R₆ is        —C(CH₃)₂.    -   R₁ is —H, R₂ is —CH₃, R₃ is —OCH₃, R₄ is —OCH₃, R₅ is —Cl and R₆        is —H,    -   R₁ is —H, R₂ is —CH₃, R₃ is —F, R₄ is —H, R₅ is —H and R₆ is —H,    -   R₁ is —Cl, R₂ is —H, R₃ is —H, R₄ is —H, R₅ is —H and R₆ is —H,    -   R₁ is —Cl, R₂ is —H, R₃ is —OCH₃, R₄ is —H, R₅ is —H and R₆ is        —H, or    -   R₁ is —H, R₂ is —CH₃, R₃ is —H, R₄ is —H, R₅ is —H and R₆ is        phenyl.

Some preferred compounds of Formula XVI include structure XVI-A, XVI-B,XVI-C, XVI-D, XVI-E, XVI-F, XVI-G, XVI-H, XVI-I, XVI-J, XVI-K, XVI-L,XVI-M or XVI-N which are set forth below in the section entitledFormulae.

Compound Source Catalog Number XVI-A. SIGMA S53,065-4 XVI-B. SYNTEC,GERMANY ST 58/4 XVI-C. SIGMA S62,937-5 XVI-D. SIGMA S50,191-3 XVI-E.SIGMA S21,210-5 XVI-F. SIGMA S21,212-1 XVI-G. SIGMA S6,965-6 XVI-H.SIGMA S6,971-0 XVI-I. SIGMA S7,002-6 XVI-J. SIGMA S21,225-3 XVI-K. SIGMAS21,234-2 XVI-L. SIGMA S21,241-5 XVI-M. SIGMA S21,243-1 XVI-N. SIGMAS63,263-5 XVI-O. SIGMA S21,212-1 XVI-P. SIGMA S38,916-1 XVI-Q. SIGMAS50,242-0 XVI-R. SIGMA S62,979-0

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XVII (SIGMA S86, 927-9).

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XVIII (RYAN SCIENTIFICE191B)

In some embodiments, the pharmaceutical compositions of the presentinvention comprise compounds having Formula XIX (SIGMA S12, 962-3).

Formulae

1-5. (canceled)
 6. A method of treating a patient suffering from aCD4-mediated disease comprising administering to said patient atherapeutically effective amount of a compound of Formula (IV), (V), or(VI),

or a pharmaceutically acceptable salt thereof.
 7. The method of claim 6,wherein said compound is a compound of formula (IV) or apharmaceutically acceptable salt thereof.
 8. The method of claim 6,wherein said compound is a compound of formula (V) or a pharmaceuticallyacceptable salt thereof.
 9. The method of claim 6, wherein said compoundis a compound of formula (VI) or a pharmaceutically acceptable saltthereof.
 10. The method of claim 6, wherein the CD4-mediated disease isselected from rheumatoid arthritis (RA), multiple sclerosis (MS),Sjogren's syndrome, sarcoidosis, insulin dependent diabetes mellitus(IDDM), autoimmune thyroiditis, reactive arthritis, ankylosingspondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis,vasculitis, Wegener's granulomatosis, Crohn's disease, ulcerativecolitis, Lupus (SLE), Grave's disease, myasthenia gravis, autoimmunehemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia,primary biliary sclerosis and pernicious anemia.
 11. A pharmaceuticalcomposition comprising a compound having the formula (IV), (V) or (VI),

or a pharmaceutically acceptable salt thereof, and a pharmaceuticalacceptable carrier or diluent.
 12. The pharmaceutical composition ofclaim 11, wherein said compound is a compound of formula (IV), or apharmaceutically acceptable salt thereof.
 13. The pharmaceuticalcomposition of claim 11, wherein said compound is a compound of formula(V), or a pharmaceutically acceptable salt thereof.
 14. Thepharmaceutical composition of claim 11, wherein said compound is acompound of formula (VI), or a pharmaceutically acceptable salt thereof.